The neurotropic flaviviruses, including West Nile virus (WNV) and Japanese Encephalitis virus (JEV) are mosquito borne viruses that cause significant morbidity and mortality worldwide. Unfortunately, current therapeutic options for treating disease associated with these viruses are limited. Recent studies by our group and others have demonstrated that microRNAs (miRNAs) play a major role in the life cycle of multiple viruses via regulation of both viral and host gene expression. miRNAs, therefore, represent potential targets for therapeutic intervention, either through enhancing or antagonizing their functions in virally infected cells. In this application, we propose to screen a library of miRNA analogs and antisense inhibitors to identify individual miRNAs that represent potential targets for therapeutic intervention. Because miRNAs are expected to function by targeting gene expression of cellular mRNAs and host-cell pathways, we will identify these targets and pathways using immunoprecipitation of the RNA-induced silencing complex (RISC), bioinformatic analysis, and experimental verification. Therefore, at the completion of the R21 portion of this proposal, we expect to have identified multiple miRNAs whose activity can be modulated to inhibit WNV and JEV replication, as well as to have characterized their mechanism of action. In the R33 portion of the proposal, fully characterized miRNAs will be evaluated for therapeutic potential of identified miRNAs in a mouse model of WNV and JEV infection.